Three Different Routes of Inoculation for Experimental Infection with Schmallenberg Virus in Sheep

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S H O R T C O M M U N I C A T I O N

Three Different Routes of Inoculation for Experimental

Infection with Schmallenberg Virus in Sheep

L. Martinelle1, A. Poskin1,2, F. Dal Pozzo1, L. Mostin3, W. Van Campe3, A. B. Cay2, N. De Regge2and C. Saegerman1

1

Research Unit of Epidemiology and Risk Analysis Applied to Veterinary Sciences (UREAR-ULg), Fundamental and Applied Research for Animals & Health (FARAH), Faculty of Veterinary Medicine, University of Liege, Liege, Belgium

2_{Veterinary and Agrochemical Research Centre (CODA-CERVA), Operational Directorate Viral Diseases, Brussels, Belgium} 3

Veterinary and Agrochemical Research Centre (CODA-CERVA), Experimental Centre, Machelen, Belgium

Keywords:

Schmallenberg; virus; sheep; inoculation; route

Correspondence:

C. Saegerman. Research Unit of Epidemiology and Risk Analysis Applied to Veterinary Sciences (UREAR-ULg), Fundamental and Applied Research for Animals & Health (FARAH), Faculty of Veterinary Medicine, University of Liege, B42, Quartier Valle´e 2, Avenue de Cureghem 7A 4000 Liege (Sart-Tilman), Belgium. Tel.: +32 4 3664579; Fax: +32 4 3664261;

E-mail: claude.saegerman@ulg.ac.be Received for publication January 10, 2015 doi:10.1111/tbed.12356

Summary

Schmallenberg virus (SBV) is an emerging Orthobunyavirus affecting European domestic ruminants. In this study, three groups of ewes (n= 3) were inoculated with 1 ml of an SBV infectious serum, via the subcutaneous (SC), intradermal (ID) or intranasal (IN) route. The ewes were monitored for 10 days and no clini-cal signs were reported. IN inoculation failed to generate any detectable RNAe-mia. SC and ID inoculation induced typical SBV RNAemia and seroconversion upon day 6 post-inoculation in 3/3 and 2/3 sheep, respectively. In all the animals that showed RNAemia, the viral genome could be detected in spleen and mesen-teric lymph nodes. Both the SC and ID routes seem suitable to properly reproduce field conditions, as comparable observations were reported regarding RNAemia, seroconversion and viral genome detection in organs.

Introduction

Schmallenberg virus (SBV; family Bunyaviridae, genus Ort-hobunyavirus) emerged in Europe in 2011 and is transmit-ted by Culicoides biting midges. It is associatransmit-ted with a mild non-specific syndrome in adult cattle, but most of the clini-cal impact is related to congenital malformations that can affect ruminant neonates following in utero infection (Her-der et al., 2012). So far, in most of the experimental infec-tions performed in host species, subcutaneous (SC) inoculation was chosen, but the intravenous (IV) (Hoff-mann et al., 2012), oral (PO) and intramuscular (IM) routes were also evaluated (Wernike et al., 2013a,b). As Cu-licoides spp. are ‘pool-feeder’, that is blood feeding follow-ing laceration of the dermis of the host, superficial tissues are in first line of contact with the pathogen. Therefore, intradermal (ID) inoculation of SBV is worth investigating. In addition, intranasal (IN) inoculation has not been tested

yet and could provide additional insights for potential SBV contact transmission. Thus, SC, ID and IN inoculations were compared based on their clinical, virological and sero-logical outcomes.

Materials and Methods

Nine ‘Mourerous’ ewes of about 1 year of age were used. The Mourerous is a middle-size rustic breed from south of France. All were serologically and virologically negative for SBV. Three isolated groups of three ewes were randomly constituted in a BSL3 facility (CODA-CERVA Machelen, Belgium). Each animal was inoculated with 1 ml of the infectious serum via the IN way (ewes IN1 to IN3) in the right nostril, or subcutaneously (SC1 to SC3) in the left axilla or for the last group, intradermally on the left side of the neck (ID1 to ID3). ID inoculation was performed using a Dermojetâ device (Akra Dermojet, Pau, France). The

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inoculum (graciously provided by Friedrich Loeffler Insti-tute, Riems, Germany) contained about 29 103_{50% tissue} culture infective dose/mL (TCID50/mL) and 7.3 9 106 RNA copies/mL of SBV S-segment. This study was approved by the Ethical Committee of the IPH-VAR (Sci-entific Institute of Public Health – Veterinary and Agro-chemical Research Centre, number of project: 121017-01, date of approval: 11 February 2013).

A daily clinical examination was performed associated with blood, faecal and nasal sampling. Ewes were eutha-nized 10 days post-infection (dpi). The extraction of RNA from organs, faeces and serum, and the detection of SBV S-segment by a one-step reverse-transcription (RT) quanti-tative PCR (RT-qPCR) were performed as described previ-ously (De Regge et al., 2013). The quantification cycle (Cq) values were converted into copy numbers (Poskin et al., 2014), and results were included only when negative extrac-tion controls and negative and positive amplificaextrac-tion controls were satisfactory.

Results and Discussion

No clinical signs were observed that could be related to an acute SBV infection in the inoculated sheep. No viral RNA

was detected in the blood, nor in the organs of the animals from the IN group. Inoculation of ID3 did not result in a detectable RNAemia. Failures to reproduce or induce infection were previously described with the same inoculum in calves (Hechinger et al., 2013) and sheep (Wernike et al., 2013b) through SC route. Thus, the lack of infection is most likely not related to the ID route itself and could be related to a random failure of infection or individual factors of resis-tance. The observed RNAemia (Figs 1a and b), in both SC and ID groups, was comparable in duration (3–5 days) and in line with previous data (Hoffmann et al., 2012; Wernike et al., 2013b; Poskin et al., 2014) following IV and SC inoc-ulations. The mean SBV genome copy numbers detected in the blood of sheep with RNAemia were not significantly different between SC and ID groups through time (two-wayANOVAwith repeated measures; group effect: P = 0.15; group–time interaction: P = 0.41). In contrast, at viraemic peak, maximum copy number was significantly higher in the ID group versus SC group (Welch’s test with unequal variance; P = 0.026).

The sporadic low-level detection of SBV genome in nasal swabs (Figs 1c and d) is highly unlikely to play a role in the epidemiology of the disease. As direct IN inoculation failed to cause RNAemia, an intermittent and low-level nasal

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Fig. 1. Reverse-transcriptase quantitative PCR results in blood (a and b) and nasal swabs (c and d) for SC and ID groups. In panels (c) and (d), no sheep had positive detection in both nostril the same day; thus, right or left nostril origin is not specified. Panels (e) and (f) show the neutralizing antibody titres per mL in SC and ID groups, respectively. ID: Intradermal and SC: Subcutaneous. [Colour figure can be viewed at wileyonlinelibrary.com].

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shedding should be of very limited impact on the further spread of the virus. SBV genome was found in faeces of ani-mals of all the three groups (Fig. 2), but only sporadically and inconstantly in animals of group IN. No SBV RNA was found in faeces of ewe ID3 that had no detectable viral

RNAemia. Mean and maximum viral RNA copy numbers and duration of detection in faeces were not significantly different between SC and ID groups (Welch’s test with unequal variance; P = 0.13, 0.12 and 0.48, respectively). In contrast with previous work (Wernike et al., 2013a), SBV genome remained detectable in faecal samples of SC and ID groups 1–2 days after the end of the positive detection in blood. The origin of the faecal positive detection, especially in IN group as no RNAemia could be detected, remains debatable. However, a gastrointestinal origin cannot be ruled out. Indeed, positive detection in IN group might be a consequence of a partial swallowing of the inoculum. Positive faeces in SC and ID groups might be related to infected cell loss or infected secretions in the gastrointesti-nal tract, further detected in faeces. Infected cells could reach gastrointestinal lumen through epithelium renewal or intestinal micro-bleeding caused by chronic parasitic infes-tation, or virus could be transferred from the blood to the bile and then to faeces. In addition, Wernike et al. (2013b) reported diarrhoea in a sheep following SBV infection, which could lead to a transient increase in intestinal perme-ability. Moreover, the 1–2 days delay between blood and negative faeces detection might be caused by the transit inertia, as mean ingesta retention time in sheep is about 48 h (Hadjigeorgiou et al., 2003). Other Bunyaviridae like hantaviruses are well known to be detected in faeces of infected animals, possibly longer than in the blood (Vouti-lainen et al., 2015).

The presence of neutralizing anti-SBV antibodies was assessed by seroneutralization (SNT, De Regge et al., 2013). Only ewes that had RNAemia seroconverted within the duration of the experiment. Seroconversion occurred between 6 and 8 dpi in SC and ID groups (Figs 1e and f). As previously reported, the decrease of SBV RNA in the blood correlated with seroconversion (Poskin et al., 2014). However, as the animals were only tested for 10 days, a later seroconversion might have been possible in ewes with-out RNAemia.

At post-mortem examination, no particular gross lesions were reported. No SBV genome could be detected in the central nervous system or genital tract. Within the same

ani-Table 1. Log copy number of SBV S-segment by weight (g) of organ for each ewe

Organ SC1 SC2 SC3 ID1 ID2 ID3

Mean by organa

Lung 0.00 5.37 0.00 5.91 0.00 0.00 5.64 (0.38) Spleen 6.46 6.70 6.72 7.69 7.44 0.00 7.00 (0.53) Mesenteric lymph node 6.69 7.30 6.75 7.91 7.51 0.00 7.23 (0.52) Submandibular lymph node 7.20 5.87 4.74 0.00 0.00 0.00 5.94 (1.23) Superficial cervical lymph node 5.54 0.00 0.00 7.09 0.00 0.00 6.32 (1.10) Mean by routea _{6.3 (0.81)} _{7.26 (0.72)}

a

Means were calculated only considering positive organs. Standard deviation is in curved brackets.

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Fig. 2. Reverse-transcriptase quantitative PCR in faeces of subcutane-ously (a), intradermally (b) and intranasally (c) inoculated animals. [Colour figure can be viewed at wileyonlinelibrary.com].

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mal, SBV RNA detection in serum was systematically associ-ated with detection in spleen and mesenteric lymph nodes.

Table 1 shows the log of copy number of SBV RNA in each positive tested organ for each animal. Considering animals that showed RNAemia, more organs were detected positive in SC group (11/15) when compared to ID group (6/10), however, this difference was not significant (Fisher’s exact test for count data= 1.79; P = 0.67). Comparing organs that were detected positive in both ID and SC groups (lung, spleen, mesenteric and superficial cervical lymph nodes), SBV RNA copy numbers were significantly higher in organs of ID group (two-wayANOVAwith repeated measures; P= 0.03).

Conclusion

IN inoculation failed to generate any detectable RNAemia. In both SC- and ID-inoculated animals, viral RNA could be detected in faeces 1–2 days longer than in the blood. The cause of this prolonged faecal viral detection is currently uncertain but not uncommon in Bunyaviridae and it might be a consequence of the digestion time in sheep. Both of the SC and ID routes seem to properly reproduce field con-ditions, as similar outcomes for parameters such as RNA-emia, seroconversion and viral genome detection in organs were found. SBV RNA copy number was significantly higher in organs of the ID group, but this result has to be interpreted with circumspection as the sample size was small and one ewe had no RNAemia at all. However, to be properly performed, ID inoculation requires the use of a regular syringe, with more care while injecting and a high risk to go SC if the animal is not perfectly calm, or specific devices such as syringe tuberculin pistol or jet injectors (like the Dermojetâ used in the current study) with the constraint of a limited volume to be injected per shot. Therefore, SC inoculation might be easier to deal with from a practical point of view.

Acknowledgements

We thank Richard Thiery and Eric Dubois from ANSES (French Agency for Food, Environmental and Occupa-tional Health & Safety, Sophia-Antipolis) for helping us in finding SBV-free animals in the field. We warmly thank all the staff of CODA-CERVA Machelen for taking care of the animals. We finally thank Martin Beer and

Bernd Hoffmann from the FLI (Riems, Germany) for providing the inoculum. This study was financially sup-ported by Federal Public Service Public Health and Safety of the Food Chain and Environment (RF12/6270) and the European Union as outlined in Council Deci-sion 2012/349/EU concerning a financial contribution by the Union for studies on Schmallenberg virus.

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